Tank-binding Kinase-1 (TBK1) (pIC50 = 6.8)

GSK8612 suppresses type I IFN production in primary human mononuclear cells and toll-like receptor (TLR)3-induced IRF3 phosphorylation in Ramos cells. GSK8612 can prevent IFNβ from being secreted in THP1 cells in reaction to dsDNA and cGAMP, which is STING's natural ligand[1].

Determination of inhibition of recombinant TBK1 [1]
The inhibition of the enzymatic activity of TBK1 by GSK8612 was determined in duplicate by Reaction Biology Corporation using their Kinase HotSpot proprietary technology. [1]

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Protocol for affinity enrichment compound competition binding assay [1]
Competition binding assays were performed as described previously by using Kinobeads and LipidKinobeads3,4 Briefly, 200 μL (1 mg protein) cell extract (see supplementary data) were pre-incubated with test compound or vehicle for 45 min at 4°C followed by incubation with kinobeads or lipid beads (7 μL beads per sample) for 1 hr at 4°C. The beads were washed with lysis buffer and eluted with 20 μL SDS sample buffer (250 mM Tris-HCl pH 7.4, 250 mM Tris Base, 20 % glycerol, 4 % SDS, 0.01 % Bromphenol blue). Proteins were digested according to a modified single pot solid-phase sample preparation (SP3) protocol. 5,6 Briefly, protein solutions were diluted to 2 % SDS and bound to S6 paramagnetic beads by addition of ethanol to a final concentration of 50%. Contaminants were removed by washing 4 times with 70% ethanol. Proteins were digested by resuspending in 0.1 mM HEPES (pH 8.5) containing TCEP, Chloracetamide, Trypsin and LysC following o/n incubation. TMT labeling was performed using the 10-plex TMT reagents, enabling relative quantification of 10 conditions in a single experiment. 4,7 The labeling reaction was performed in 40 mM triethylammoniumbicarbonate, pH 8.5, at 22°C and quenched with glycine. Labeled peptide extracts were combined to a single sample per experiment. Samples were dried in vacuo and resuspended in 0.05% trifluoroacetic acid in water. 30% of the sample was injected into an Ultimate3000 nanoRLSC coupled to a Q Exactive. Peptides were separated on custom-made 35 cm × 100 μm (ID) reversed-phase columns (ReproSil-pur C18-AQ, 1.9 µm) at 55°C. Gradient elution was performed from 2% acetonitrile to 40% acetonitrile in 0.1% formic acid and 3.5% DMSO over 3.3 h. Samples were online injected into Q-Exactive mass spectrometers operating with a data-dependent top 10 method. MS spectra were acquired by using 70.000 resolution and an ion target of 3E6. Higher energy collisional dissociation (HCD) scans were performed with 35% NCE at 35.000 resolution (at m/z 200), and the ion target settings was set to 2E5 so as to avoid coalescence. 7 The instruments were operated with Tune 2.9 and Xcalibur 4.1. [1]

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Determination of physical chemical properties [1]
CLND solubility, CHROM LogD, Artificial membrane permeability and human serum albumin binding were all determined according to the procedures reported by Diaz et al.

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Protocol for determination of the unbound compound fraction in blood [1]
A rapid equilibrium dialysis technique using 48-well rapid equlibrium dialysis plates with LC-MS/MS analysis was used to determine the unbound fraction of GSK8612 in rat, mouse and human blood. Concentrations of 200 ng/ml and 1000 mg/ml of compounds were spiked into blood (0.5% final concentration of organic solvent). Phosphate buffered saline (100 mM sodium phosphate + 150 mM sodium chloride pH 6.9-7.2) was used as the dialysis buffer. 100 µL of spiked blood was allowed to equilbrate with 300 µL of dialysis buffer (n = 6) by incubation at 37oC for 4 h in rapid equilibrium dialysis plates. Samples were analysed by LC-MS/MS using Labetalol as the internal standard and the unbound compound fraction in buffer was calculated.

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Protocol for the determination of microsomal stability [1]
The study was carried out by the CRO Cyprotex. Pooled mouse, rat or human liver microsomes (protein concentration 0.5 mg/mL) in phosphate buffer (pH 7.4) with NADPH (1 mM) were incubated with 0.5 µM of GSK8612 (0.25% DMSO) at 37°C for total of 45 minutes. Aliquots (50 µL) of each experiment were taken at 0, 5, 15, 30 and 45 min and the reactions were stopped by the addition of 100 µL methanol containing internal standard. These samples were centrifuged at 2,500 rpm for 20 min at 4°C to precipitate the proteins and the samples were then analysed by LC-MS/MS. Control samples were also run under same conditions but with no NADPH added.

Inhibition of phosphorylation of IRF3 in Ramos cells [1]
Ramos cells (ATCC, 106 /well) were exposed to GSK8612 for 60 min in cell culture media (RPMI1640) containing 2% fetal bovine serum and then stimulated with poly(I:C) (30 µg/mL) for 120 min at 37°C, 5% CO2. Cells were then collected and washed once with ice-cold DPBS. The cells were lysed in 50 mM Tris-HCl (pH 7.4) containing 5% glycerol, 1.5 mM MgCl2, 20 mM NaCl, 1 mM Na3VO4, 0.8% (v/v) IGEPAL™-CA630, 50 nM Calyculin A, phosphatase inhibitors mix, protease inhibitors mix (aprotinin, bestatin, leupeptin, pepstatin, phosphoramidon), 25 mM NaF, 1 mM dithiothreitol. Cell debris were removed by centrifugation and the soluble proteins were denatured with NuPAGE™ LDS Sample Buffer supplemented with 50 mM dithiothreitol). The samples were analysis by Western blot using 4-10% polyacrylamide gel and antibodies. The relative phosphorylation of IRF3 was analysed in SDS lysates by Western blot. Band intensities were quantified using a LICOR ODYSSEY™ scanner. The normalised phospho-IRF3 signal is displayed as percent inhibition, with unstimulated cells giving the minimum signal and vehicle-treated poly(I:C)-stimulated cells giving the maximum signal. The pIC50 was derived from a four-parameter sigmoidal curve fit constrained to top (100%) using GraphPad Prism software (V7).

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Inhibition of IFNα secretion in human PBMCs [1]
Peripheral blood mononuclear cells (PBMC) were isolated from human whole blood by density centrifugation using a Ficoll-based medium). PBMCs were seeded in 96-well plates at 50,000 cells per well in 50 µL media (RPMI1640) with 10% fetal bovine serum. Cells were incubated with GSK8612 or vehicle (DMSO 0.2%) for 1 h at 37°C, 5% CO2. The cells were then stimulated with 100 μg/ml poly(I:C) for 16 h at 37°C, 5% CO2. The supernatant was collected and analysed by multiplex Cytometric Bead Array (CBA) Flex Sets for secreted IFNα. Percent inhibiton of IFNα secretion was calculated based on the mean fluoresence intensities (MFI) measured by flow cytometry.

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Inhibition of IFN [1]
The secretion in human THP-1 THP-1 cells (ATCC) were plated in 96-well tissue culture plates at 100,000 cells per well in 100 µL media (RPMI-140, 10 % heat-inactivated FBS, 1 % penicillin/streptomycin/amphotericin) followed by 45 min incubation at 37°C with increasing concentrations of GSK8612 (0.003-10 µM). Cells were stimulated with 10 µL Bacmam virus (7.32 X 108 pfu/mL) or 10 µL of 600 µg/mL cGAMP solution (in water). Levels of secreted IFN were measured from cell supernatants following a 20 hour incubation by electrochemiluminescence using a MESO Sector S 600 platform following the manufacturer’s instructions for the MSD 96-well MULTI-ARRAY Human IFN-b assay (K111ADB-2). Data from three technical replicate experiments were averaged, plotted as a function of GSK8612 concentration in Graphpad Prism 4.0 and fit to model of inhibition to determine the IC50.

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Cell culture and lysate preparation for chemoproteomic analysis [1]
HEK293 (ACC-305, DSMZ), 1 K-562 (ACC-10, DSMZ),2 HepG2 (ATCC), HeLa (ATCC), Jurkat (ATCC), and Ramos cell lines were maintained in standard cell culture media made of DMEM or RPMI-1640 supplemented with 10% fetal calf serum without antibiotics. Human placenta was sourced from Biopredic and ABS. Mixed HEK293, K-562, HepG2 and human placenta cell extracts and mixed HeLa, Jurkat, and K-562 cell extracts were prepared as described. 3 For the analysis of different kinase activation states, Ramos cells were either left untreated or were stimulated with 50 nM Calyculin A and 0.1% DMSO for 30 min at 37°C, 5% CO2, followied by lysis steps as described above for other lysates.

[1]. Discovery of GSK8612, a Highly Selective and Potent TBK1 Inhibitor. ACS Med Chem Lett. 2019 Mar 11;10(5):780-785.

The serine/threonine protein kinase TBK1 (Tank-binding Kinase-1) is a noncanonical member of the IkB kinase (IKK) family. This kinase regulates signaling pathways in innate immunity, oncogenesis, energy homeostasis, autophagy, and neuroinflammation. Herein, we report the discovery and characterization of a novel potent and highly selective TBK1 inhibitor, GSK8612. In cellular assays, this small molecule inhibited toll-like receptor (TLR)3-induced interferon regulatory factor (IRF)3 phosphorylation in Ramos cells and type I interferon (IFN) secretion in primary human mononuclear cells. In THP1 cells, GSK8612 was able to inhibit secretion of interferon beta (IFNβ) in response to dsDNA and cGAMP, the natural ligand for STING. GSK8612 is a TBK1 small molecule inhibitor displaying an excellent selectivity profile and therefore represents an ideal probe to further dissect the biology of TBK1 in models of immunity, neuroinflammation, obesity, or cancer.[1]
In summary, the biological activity of GSK8612 was demonstrated, resulting in inhibition of IRF3 phosphorylation in Ramos cells, IFNα secretion from human PBMCs, and IFNβ secretion from THP-1 cells with low micromolar potency. GSK8612 is a highly selective TBK1 inhibitor, thus representing an ideal tool to further dissect the physiological roles of TBK1 in biological models of immunity, neuroinflammation, obesity, and cancer.[1]

C17H17BRF3N7O2S

520.3268

519.029

C, 39.24; H, 3.29; Br, 15.36; F, 10.95; N, 18.84; O, 6.15; S, 6.16

2361659-62-1

137553174

White to off-white solid powder

1.7±0.1 g/cm3

671.0±65.0 °C at 760 mmHg

359.6±34.3 °C

0.0±2.1 mmHg at 25°C

1.674

1.77

3

11

7

31

688

0

FFPHMUIGESPOTK-UHFFFAOYSA-N

InChI=1S/C17H17BrF3N7O2S/c1-10-14(8-28(27-10)9-17(19,20)21)25-16-24-7-13(18)15(26-16)23-6-11-2-4-12(5-3-11)31(22,29)30/h2-5,7-8H,6,9H2,1H3,(H2,22,29,30)(H2,23,24,25,26)

4-((5-bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide

GSK-8612; GSK 8612; GSK8612; 2361659-62-1; 4-(((5-Bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)amino)methyl)benzenesulfonamide; CHEMBL4446892; 4-[[[5-bromo-2-[[3-methyl-1-(2,2,2-trifluoroethyl)pyrazol-4-yl]amino]pyrimidin-4-yl]amino]methyl]benzenesulfonamide; 4-((5-Bromo-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide; GSK8612;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 125 mg/mL (~240.23 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)